Some molecular biology methods demand enzymes that can be activated in a controlled manner while being already contained in the reaction. This allows starting sequential enzymatic procedures in the same reaction container at pre-defined time points or avoiding the generation of undesired side products due to premature start of the enzymatic reaction. For the use of chemically modified thermostable enzymes, two such modifications have been described for thermostable DNA polymerases (see U.S. Pat. Nos. 5,677,152; 5,773,258; and 6,183,998, which are incorporated by reference herein in their entireties). Such modifications can also be employed for other thermostable enzymes since amino acids become modified by these chemicals that are contained in all enzymes and are often involved in the catalytic centre of enzymes such as amino acids carrying a free NH2-group. Furthermore, U.S. Pat. No. 6,183,998 teaches the crosslinking of enzyme molecules by aldehydes, which is generically suitable to modify any thermostable enzyme. The activity of such chemically modified enzymes becomes restored by a heat incubation step that breaks the covalent bonds to the amino acid residues thereby restoring the catalytic activity of the enzyme. However, depending on the inactivation procedure or the modifier concentration chosen, enzyme activity is restored only at a slow rate.
The inventors have discovered that nitrogen-containing compounds surprisingly enhance enzyme activity of chemically modified enzymes while they have no effect on the unmodified enzyme. Thus, this invention describes for the first time a mechanism that enhances the reactivation of chemically modified enzymes, which is suitable in many applications, e.g., to increase speed of the enzymatic reaction.